MOLECULAR MEDICINE REPORTS 12: 5494-5500, 2015

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Deglucose chikusetsusaponin IVa isolated from Rhizoma Panacis Majoris induces apoptosis in human HepG2 hepatoma cells XIAOMEI SONG*, WEI WANG*, XIN ZHANG, YI JIANG, XINJIE YANG, CHONG DENG, ZHENGGANG YUE and ZHISHU TANG Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization, Shaanxi Province Key Laboratory of New Drugs and Chinese Medicine Foundation Research, Shaanxi Rheumatism and Tumor Center of TCM Engineering Technology Research, School of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, P.R. China Received September 3, 2014; Accepted May 19, 2015 DOI: 10.3892/mmr.2015.4035 Abstract. Deglucose chikusetsusaponin IVa (DCIVa), isolated from Rhizoma Panacis Majoris, a widely used traditional Chinese medicine, is a type of oleanane triterpenoids. Various previous studies have demonstrated that oleanane triterpenoids exhibit cytotoxic activity against various types of cancer cells. However, whether DCIVa exerts an antitumor effect remains to be elucidated. The present study aimed to assess the effect of DCIVa on cancer cells using the HepG2 hepatocellular carcinoma cell line and determine the underlying mechanism. Using an MTT assay, it was demonstrated that DCIVa inhibited cell growth and viability in a dose‑ and time‑dependent manner. Typical apoptotic features, including chromatin condensation and margination at the nuclear periphery, and apoptotic body formation were induced by DCIVa and were detected by transmission electron microscopy. In addition, nuclear condensation and fragmentation were also observed by Hoechst 33258 staining. Furthermore,

Correspondence to: Dr Zhishu Tang or Dr Xiaomei Song, Shaanxi

Collaborative Innovation Center of Chinese Medicinal Resource Industrialization, Shaanxi Province Key Laboratory of New Drugs and Chinese Medicine Foundation Research, Shaanxi Rheumatism and Tumor Center of TCM Engineering Technology Research, School of Pharmacy, Shaanxi University of Chinese Medicine, Century Avenue, Xianyang, Shaanxi 712046, P.R. China E‑mail: [email protected] E‑mail: [email protected]

Abbreviations: DCIVa, deglucose chikusetsusaponin IVa; TCM, traditional Chinese medicine; FBS, fetal bovine serum; MTT, 3‑(4,5‑dimethylthi‑azol‑2‑yl)‑2,5‑diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide; PI, propidium iodide; HRP, horseradish peroxidase *

Contributed equally

Key words: Rhizoma Panacis Majoris, antitumor, deglucose

chikusetsusaponin IVa, cytotoxic activity, cell apoptosis, cell cycle arrest

flow cytometric analysis revealed that DCIVa increased cell apoptosis and G2/M cell cycle arrest dose‑dependently. Western blot analysis further demonstrated that DCIVa upregulated the expression of the pro‑apoptotic protein, Bax, and downregulated the expression of the anti‑apoptotic protein, Bcl‑2. In conclusion, the present study demonstrated for the first time, to the best of our knowledge, that DCIVa exerts potent cytotoxic effects on HepG2 cells through induction of cell apoptosis and cell cycle arrest, and may be utilized as a potential anticancer agent. Introduction Traditional Chinese medicine (TCM), as a complementary and alternative medicine, has been widely used in China for thousands of years (1). From ancient to modern times in China, TCM has been critical in the prevention and treatment of several diseases prior to western medicine being introduced (2). The benefits of TCM are gradually being recognized worldwide, which may aid in the development of novels drugs for the treatment of various types of disease. Rhizoma Panacis Majoris, also named Zhu Zi Shen or Kou Zi Qi in Chinese, has been widely used for the treatment of several diseases due to its various pharmacological activities. In China, Rhizoma Panacis Majoris is predominantly located in the southwest area, including Shaanxi, Gansu, Ningxia, Henan, Hubei and Yunnan provinces, at an altitude of 1200‑4000 m in the valley of broad‑leaved forest (3). Modern chemical investigations have demonstrated that Rhizoma Panacis Majoris predominantly contains saponins, naphtha and organic acids. (4,5). It has been revealed that Rhizoma Panacis Majoris possesses several pharmacological activities. For example, the total saponins of Rhizoma Panacis Majoris have been demonstrated to increase the proliferation of T lymphocytes induced by Sword bean A and Phytohemagglutinin (6). The water extract exhibits anti‑inflammatory and analgesic effects in mice (7), and protects the mice against acute cerebral ischemia‑reperfusion injury (8). Furthermore, Rhizoma Panacis Majoris was revealed to be capable of reducing the toxicity of chemotherapeutic drugs (9) and exerting an antitumor effect (10,11).

SONG et al: DEGLUCOSE CHIKUSETSUSAPONIN IVA INDUCES CELL APOPTOSIS

Deglucose chikusetsusaponin IVa (DCIVa), also termed Oleanolic acid‑3‑O‑β‑D‑pyran glucuronic acid glycoside, is also isolated from the extract of Rhizoma Panacis Majoris (12). DCIVa has been revealed to exert an anti‑arrhythmic effect (13) and protect against hypoxia/reoxygenation‑induced injury in myocardial cells (14). DCIVa is a member of the family of oleanane triterpenoids, which has been reported to have numerous pharmacological activities, including cytotoxic activity against various cancer cells, anti‑inflammatory activity, prevention of dental caries and induction of gentamicin nephrotoxicity (15‑18). However, whether DCIVa exerts an antitumor effect remains to be elucidated. The present study aimed to investigate the antitumor effect and the underlying mechanism of DCIVa using HepG2 hepatocellular carcinoma cells. Materials and methods Reagents. Fetal bovine serum (FBS) was obtained from Minhai Biotechnology Development (Beijing, China) and RPMI‑1640 medium was obtained from Gibco‑BRL (Gaithersburg, MD, USA). 3‑(4,5‑dimethylthi‑azol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33258 and propidium iodide (PI) were purchased from Sigma‑Aldrich (St. Louis, MO, USA). Cell culture. The HepG2 hepatocellular carcinoma cells were obtained from The Fourth Military Medical University (Xian, China). The cells were cultured in RPMI‑1640 medium supplemented with 10% FBS, containing 100 µg/ml streptomycin, 100 U/ml penicillin and 0.03% L‑glutamine (all obtained from Sigma‑Aldrich) and were maintained at 37˚C with 5% CO2 in a humidified atmosphere. MTT assay. The HepG2 cells were seeded in 96‑well tissue culture plates (Nunc, Roskilde, Denmark) at a density of 1x104 cells/well. Following a 12 h incubation, the cells were treated with or without DCIVa at a concentration of 0.02, 0.04, 0.06, 0.08 or 0.1 µmol/ml, and incubated for 24, 48 or 72 h. Following incubation, MTT (5 mg/ml in phosphate‑buffered saline; PBS) was added (200 µl/well) and incubated for an additional 4 h. DMSO (150 µl/well) was subsequently added to dissolve the formazan product for 15 min. The absorbance at 490 nm was measured using an ELISA reader (Bio‑Tek, Winooski, VT, USA). The experiment was performed five times. The data are representative of three independent experiments. The cell growth inhibition rate was calculated as follows: Cell growth inhibition (%) = HepG2control  ‑ HepG2DCIVa / HepG2control x 100. Observation of morphological changes. The HepG2 cells were seeded into 6‑well culture plates at a density of 1x104 cells/well. Following a 12 h incubation, the cells were treated with different concentrations of DCIVa for 24 h. The cellular morphology was observed using an inverted microscope (AE21; Motic, Xiamen, China). Transmission electron microscopy. The HepG2 cells were seeded into culture flasks at a density of 1x106 cells/flask. Following a 12 h incubation, the cells were treated with or

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without 0.06 µmol/ml DCIVa for 24 h. The cells were subsequently collected and fixed with fixative. The cell samples were processed into ultra‑thin sections in the College of Medicine of Xian Jiaotong University (Xian, China). The ultra‑thin sections were examined using a H600 transmission electron microscope (Hitachi, Tokyo Japan). Hochest 33258 staining. The cells were treated with 0, 0.06 or 0.1 µmol/ml DCIVa for 24 h and washed with ice‑cold PBS twice prior to fixation with 3 ml ethyl alcohol for 30 min at room temperature. The fixative was discarded and the cells were washed three times with ice‑cold PBS. Hoechst 33258 (5 mg/l) was added to the cells and incubated for 45 min in the dark. The cells were washed twice with ice‑cold PBS followed by visualization under a Leica AF6000 fluorescence microscope (Leica, Wetzlar, Germany). Flow cytometric analysis. For cell cycle analysis, following treatment with different concentrations of DCIVa, the cells were harvested and washed with ice‑cold PBS. The cells were subsequently fixed in 70% ethanol and maintained at 4˚C for at least 12 h. The cell pellets were obtained by centrifugation at 2,000 x g for 10 min and then stained with a fluorescent probe solution, containing 50 µg/ml PI and 1 mg/ml DNase‑free RNaseA in PBS on ice in the dark for 30 min. The DNA fluorescence of the PI‑stained cells was determined using FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Analysis of cell apoptosis was performed using an Annexin V/PI apoptosis detection method (Beyotime Institute of Biotechnology, Haimen, China). Briefly, the cells were collected and washed with ice‑cold PBS followed by resuspension in binding buffer. The cells were incubated with 10 µl Annexin V stock solution for 30 min at 4˚C. A total of 5 µl PI was subsequently added and the cells were incubated for 5 min. Finally, cells were analyzed by flow cytometry. Western blot analysis. The HepG2 cells were cultured for 24 h and the old culture medium was replaced with fresh medium without phenol red and FBS. Following incubation for a further 24 h, the cells were treated with or without DCIVa at the provided concentrations and incubated for 24 h. The adherent and floating cells were harvested and washed with PBS. The cell pellets were obtained by centrifugation at 2,000 x g for 10 min and then resuspended in lysis buffer, containing 50 mM HEPES (pH 7.4), 1% Triton X‑100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM PMSF, 10 mg/l aprotinin and 10 mg/l leupeptin, and lysed at 4˚C for 60 min. Following centrifugation at 13,000 x g for 15 min, the protein concentration in the supernatant was determined using a Bradford assay (Beyotime Institute of Biotechnology). A total of 20 µg protein lysate was separated by electrophoresis on 12% SDS‑polyacrylamide gels (Sangon Biotech Co., Ltd., Shanghai, China) and transferred onto a nitrocellulose membrane (Bio‑Rad, Hercules, CA, USA). The membranes were soaked in blocking buffer (5% non‑fat milk) at 37˚C for 1 h. The membranes were incubated with polyclonal rabbit anti‑human Bcl‑2 antibody (1:500; cat. no. sc-492) monoclonal mouse anti‑human Bax antibody (1:1,000; sc‑20067)and polyclonal rabbit anti‑human β‑actin antibody

MOLECULAR MEDICINE REPORTS 12: 5494-5500, 2015

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Figure 1. Detection of the cytotoxicity of DCIVa on the HepG2 cells. (A) The chemical structure of DCIVa. (B) The cells were treated with different concentrations of DCIVa (0.02, 0.04, 0.06, 0.08 or 0.1 µM) for 24, 48 or 72 h. The inhibitory ratio was determined by an MTT assay. (C) The cellular morphological changes were observed by inverted microscopy following treatment with (Ca) 0.1, (Cb) 0.08, (Cc) 0.06, (Cd) 0.04, (Ce) 0.02 and (Cf) 0 µmol/ml). DCIVa, deglucose chikusetsusaponin IVa.

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Figure 2. Detection of cell ultrastructure by transmission electron microscopy. (A‑C) The control group treated with 0 µmol/ml DCIVa and (D‑F) the experimental cells were treated with 0.06 µmol/ml DCIVa (scale bar of A and D, 5 µm; scale bar of B and E, 2 µm; scale bar of C and F, 0.5 µm). DCIVa, deglucose chikusetsusaponin IVa.

(1:800; sc‑130656)obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) at 4˚C overnight. The membrane was subsequently incubated with anti‑rabbit or anti‑mouse IgG conjugated with horseradish peroxidase (HRP) for 1 h. The proteins were detected using 3,3'‑diaminobenzidine tetrahydrochloride as the HRP substrate. Statistical analysis. All data are expressed as the mean ± standard deviation of at least three independent experiments. Statistical comparisons between or among groups were analyzed by two‑tailed Student's t‑test or one‑way analysis of variance, followed by Bonferroni post hoc. Statistical analysis was performed using SPSS version 11.5 (SPSS, Inc., Chicago, IL, USA). P

Deglucose chikusetsusaponin IVa isolated from Rhizoma Panacis Majoris induces apoptosis in human HepG2 hepatoma cells.

Deglucose chikusetsusaponin IVa (DCIVa), isolated from Rhizoma Panacis Majoris, a widely used traditional Chinese medicine, is a type of oleanane trit...
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